Cancer Research Cell Death Mechanisms and Cancer Therapy Protein Translation and Cancer
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Yasuda, M.
Right arrow Articles by Yasumoto, K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Yasuda, M.
Right arrow Articles by Yasumoto, K.
[Cancer Research 61, 2022-2030, March 1, 2001]
© 2001 American Association for Cancer Research

Immunology

Stimulation of ß1 Integrin Down-Regulates ICAM-1 Expression and ICAM-1-dependent Adhesion of Lung Cancer Cells through Focal Adhesion Kinase1

Manabu Yasuda, Yoshiya Tanaka2, Masahito Tamura, Koichi Fujii, Masakazu Sugaya, Tomoko So, Mitsuhiro Takenoyama and Kosei Yasumoto

Second Department of Surgery [M. Y., M. S., T. S., M. Tak., K. Y.], First Department of Internal Medicine [Y. T., K. F.], and Kidney Center [M. Tam.], School of Medicine, University of Occupational and Environmental Health, Japan, Kitakyushu 807-8555, Japan


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Adhesion molecules are involved in intracellular signaling in various physiological and pathological processes including metastasis and growth of tumor cells. Tumor cells interact with various host cells as well as with extracellular matrices through certain adhesion molecules such as integrins. We here propose that stimulation of ß1 integrin reduces intercellular adhesion molecule (ICAM)-1-mediated interaction of lung cancer cells with CTLs. This concept is based on the following findings: (a) engagement of ß1 integrins on certain lung cancer cells by a specific antibody or by ligand matrices such as fibronectin and collagen markedly reduced ICAM-1 expression on the cell surface and induced sICAM-1; (b) down-regulation of ICAM-1 by stimulation of ß1 integrins was abrogated by tyrosine kinase inhibitors or by transfection of dominant negative truncations of focal adhesion kinase (FAK); (c) engagement of ß1 integrins also reduced ICAM-1-dependent adhesion of lung cancer cells to T cells, a process completely inhibited by tyrosine kinase inhibitors and by transfection of dominant negative forms of FAK; and (d) stimulation of ß1 integrins prevented killing of lung cancer cells by autologous CTLs. In malignant tumors, cancer cells, including lung cancer cells, are surrounded by extracellular matrix proteins such as fibronectin and collagen. This suggests that the engagement of ß1 integrins by matrix proteins potentially occurs in cancer cells in vivo and that continuous stimulation via ß1 integrins reduces ICAM-1-expression, ICAM-1-mediated adhesion of cancer cells to CTLs and their killing by CTLs. Our results suggest that such processes can lead to the escape of lung cancer cells in vivo from immunological surveillance.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Metastasis, the spread of cells from the primary neoplasm to distant sites and their growth at that location, is the most fearsome aspect of cancer. Despite significant improvements in early diagnosis, surgical techniques, general patient care, and local and systemic adjuvant therapies, most deaths from cancer are attributable to metastases that are resistant to conventional therapies. During the metastatic cascade, tumor cells interact with various host cells as well as with extracellular matrices and basement membrane components including laminin, fibronectin, and type I collagen through certain adhesion molecules such as integrins (1, 2, 3) . Such adhesive interaction may lead to the enhancement of survival, arrest, or invasiveness of tumor cells and is one of the most important events in the metastatic process (4, 5, 6, 7) .

Adhesion molecules are involved in intracellular signaling in a variety of physiological and pathological processes including metastasis and tumor growth. Recent studies have indicated that certain adhesion molecules serve not only as adhesive substances but also regulate several cellular functions by influencing signaling, designated "outside-to-in signaling." The many better-known molecules are integrins ß1 and ß2 and CD28, which induce costimulatory signals in the binding of T cells to antigen-presenting cells via multiple cellular signaling molecules, including FAKs,3 resulting in cell activation and cytokine production (8) . Cell adhesion to matrices is primarily mediated by integrins, cell-surface receptors that comprise an expanding family of transmembrane heterodimers of {alpha} and ß subunits (9, 10, 11, 12) . Several studies have demonstrated that integrins play a key role in the malignant behavior of neoplastic cells (13, 14 , 16, 17, 18) . Interaction of integrins with their protein ligands increases tyrosine phoshorylation and triggers the assembly of cytoskeletal proteins, signaling enzymes, and their substrates into membrane-substratum junctions referred to as focal adhesions (19, 20, 21, 22) .

The expression and function of adhesion molecules are regulated through intracellular signaling induced by several cellular stimuli, a process designated "inside-to-out signaling." Among these molecules, the expression of ICAM-1 is tightly regulated by locally produced inflammatory cytokines such as IL-1ß, tumor necrosis factor {alpha}, IL-6, and IFN-{gamma} (23 , 24) . The ICAM-1/LFA-1 pathway regulates important cell-cell interactions such as leukocyte adhesion and migration, especially the killing of tumor cells by natural killer cells and CTLs (25 , 26) . Although various tumor cells are known to highly express ICAM-1, a potent ligand for LFA-1 on CTL in vitro, many tumor cells remain viable against killing by CTL in vivo. However, the regulation of ICAM-1 expression on cancer cells is still unclear.

In the present study, we examined the role of ß1 integrin-mediated signaling in the regulation of cell surface adhesion molecules using lung cancer cells. Our results showed that the engagement of ß1 integrin by a specific Ab or ligand matrices reduced ICAM-1 expression through tyrosine kinases and FAK, which subsequently resulted in reduced adhesion of lung cancer cells to T cells and which protected cancer cells from CTL-mediated cytotoxicity.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
The study protocol was approved by the Human Ethics Review Committee of the University of Occupational and Environmental Health, Japan ( Kitak yushu, Japan), and a signed consent form was obtained from each subject prior to taking tissue samples used in the present study.

Tumor Cell Lines.
Eleven human lung cancer cell clones were used in the present study; A904L, C831L (lung large cell carcinoma), A110L, C422L (lung adenocarcinoma), and B1203L (lung squamous cell carcinoma). These cell lines were established in our laboratory as described previously (27, 28, 29, 30, 31) . PC-9 and A549 were derived from lung adenocarcinoma (30) and QG56 was derived from lung squamous cell carcinoma (29) . PC-1, PC-6 (32) , and QG90 (28) were derived from lung small cell carcinoma. All of the cell lines were grown in RPMI 1640 (Nissui, Tokyo, Japan) with 10% FCS (Bio-Pro, Karlsruhe, Germany).

Induction of CTLs.
RLNLs from lung cancer patients were harvested at the time of surgery as described previously (30) . RLNLs were stimulated with solid-phase anti-CD3 mAb (Ortho Pharmaceutical Corporation, Raritan, NJ) for 48 h and were expanded in RPMI 1640 containing 10% FCS and 50 Japan reference units (JRU)/ml recombinant human IL-2 (Takeda Chemical Co, Osaka, Japan) for 14 days (33) . Subsequently, RLNLs were stimulated with irradiated autologous tumor cell lines weekly for 2–4 weeks (30) .

Antibodies and Reagents.
The following mAbs were used as purified immunoglobulins in cell surface analyses and functional assays: CD11a (LFA-1{alpha}) mAb TS1/22; MHC class I mAb W6/32; anti-MHC class II mAb L227; antiglycophorin mAb 10F7 [from American Type Culture Collection (ATCC), Rockville, MD]; CD29 (ß1 integrin) mAb mAb13 (kindly donated by Dr. K. M. Yamada, NIH, Bethesda, MD); CD29 mAb TS2/16 (ATCC); CD29 mAb Lia1/2 (Beckman-Coulter, Tokyo, Japan); CD29 mAb MAR4 (Becton Dickinson, San Jose, CA); CD44 mAb NIH44–1 (from Dr. S. Shaw, NIH, Bethesda, MD); CD54 (ICAM-1) mAb HA58; control murine IgG1 (Becton Dickinson); CD95 (Fas) mAb UB2 (Medical and Biological Laboratories Co., Nagoya, Japan); and CD106 (VCAM-1) mAb 2G7 (from Dr. W. Newman, Otsuka America, Rockville, MD). A human wild-type FAK expression plasmid VSV-FAK, a human FAT expression plasmid VSV-FAT, and a human FRNK expression plasmid VSV-FRNK were constructed as described previously (34 , 35) . The anti-FAK Ab A-17 (Santa Cruz, Santa Cruz, CA), anti-VSV glycoprotein mAb (Sigma, St. Louis, MO), anti-FAK (pY397) phos phor yla tion-specific Ab (Biosource, Camarillo, CA) were used for Western blotting. Multiple inhibitors of intracytoplasmic signaling including wortmannin (Wako Pure Chemical, Osaka, Japan), a PI-3-kinase inhibitor; H7 and staurosporine (Seikagaku, Tokyo, Japan), C-kinase inhibitors; herbimycin A (Sigma) and genistein (Calbiochem, San Diego, CA), tyrosine kinase inhibitors were applied to each assay system; and all of the reagents were used at the indicated concentrations. At these concentrations, none of these inhibitors produced cytotoxic effects on lung cancer cells, as confirmed by trypan blue staining.

Stimulation of Lung Cancer Cells by ß1 Integrin Using mAbs and Substrates.
After cells were cultured to subconfluence, the medium was changed to RPMI 1640 containing 1% FCS at the day before assay. The obtained cells were then stimulated with anti-ß1 integrin mAbs such as mAb13 and control mAbs (1 µg/ml) in RPMI 1640 without FCS for the indicated duration at 37°C. Signal inhibitors were added at the indicated concentration for 30-min incubation, prior to cell stimulation. After three washes, cells were stimulated with mAbs as described above and were incubated in RPMI 1640 without FCS at 37°C for the indicated duration. Purified fibronectin (10 µg/ml), collagen type I (10 µg/ml), and control HSA (10 µg/ml; Welfide, Osaka, Japan) were applied to 6-well plastic plates in Ca/Mg-free PBS at 4°C overnight. Binding sites on plastic were subsequently blocked with Ca/Mg-free PBS/2.5% HSA for 2–3 h at 37°C to reduce nonspecific attachment of the cells. Subsequently, plates were washed three times with PBS, and the cells were added to each well as described above and were incubated in RPMI 1640 without FCS at 37°C for 24 h on the indicated duration in Fig. 2CCitation . After the incubation, the plates were washed with PBS twice, were treated with trypsin for 1 min at 37°C and were quickly refilled with RPMI 1640 containing 10% FCS to quit trypsinization. After harvesting from the wells, the obtained cells were washed with PBS and were settled in media suitable for the following experiments.



View larger version (34K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 2. Stimulation of ß1 integrin reduces ICAM-1 expression on A904L cells. A, (a), down-regulation of ICAM-1, but not VCAM-1, Fas, or MHC class I, by ß1 integrins on A904L cells; (b) B1203L cells also show high expression of ß1 integrins, but stimulation of this molecule did not change the expression of ICAM-1 on the cells. A904L cells and B1203L cells were stimulated with ({blacksquare}) or without ({square}) 1 µg/ml of ß1 mAb mAb13 for 24 h, and the expression of ICAM-1, VCAM-1, Fas, and MHC class I antigen was analyzed by FACScan. B, down-regulation of ICAM-1 by ß1 integrins, but not control molecules on A904L cells. A904L cells were stimulated with 1 µg/ml of ß1 mAb mAb13, MHC class I mAb W6/32, CD44 mAb NIH44–1, and VCAM-1 mAb 2G7 as a control for 24 h. After incubation, ICAM-1 expression was analyzed by FACScan. C, kinetic of ICAM-1 reduction by ß1 integrins on lung cancer cell clones. A904L and A110L cells were stimulated with or without 1 µg/ml of ß1 mAb mAb13 for the indicated, duration and the expression of ICAM-1 was analyzed by FACScan. Each bar, the number of molecules expressed per cell, calculated using standard QIFKIT beads. Representative data of five similar experiments are shown.

 
Transfection of Plasmid.
A human wild-type FAK expression plasmid VSV-FAK, a FAT expression plasmid VSV-FAT, and a FRNK expression plasmid VSV-FRNK were introduced into lung cancer cells using a cationic liposome-mediated transfection method. Plasmid dissolved (2.5 µg) in 100 µl of serum-free medium, OPTI-MEM (Life Technologies, Gaithersburg, MD) were mixed with 5 µl of lipofectin reagent (LipofectAMINE2000 or LipofectAMINEplus, Life Technologies) in the same volume of OPTI-MEM and incubated for 15 min at room temperature. The plasmids and liposome complex were added to lung cancer cells plated in a 6-well culture dish, incubated for 3 h in OPTI-MEM, and then replaced with RPMI 1640 containing 10% FCS for 24 h. The expression of VSV-FAK, VSV-FAT, and VSV-FRNK in A904L cells was confirmed by Western blotting and flow cytometric analysis using anti-VSV Ab after their transfection. High expression of the reporter VSV was observed in Western blotting, and more than 75% of the cells were transfected by intracellular flow cytometric analysis (data not shown).

Western Blotting.
After transfection of cells expressing the various constructs, the cells (5 x 105) were stimulated for 1 h with mAb13 mAb. The cells were washed twice with ice-cold PBS and solubilized in radioimmunoprecipitation lysis buffer (Boehringer Mannheim, Mannheim, Germany). The homogenates were clarified by centrifugation at 12,000 x g for 15 min at 4°C, protein concentrations were determined using a protein microassay (Bio-Rad, Tokyo, Japan), and samples were adjusted to equal protein concentration and volume. Each sample was electrophoresed on 4–12% polyacrylamide gels and electrophoretically transferred to polyvinylidene difluoride membrane (Bio-Rad) for 1.5 h at 250 mA. The filters were incubated with blocking buffer {5% nonfat dry milk; alternatively, 5% BSA for antiphosphotyrosine Ab in T-TBS [150 mM NaCl, 50 mM Tris-HCl, and 0.1% Tween 20 (pH7.4)]} for 1 h and then incubated for 1 h with antibodies. Blots were visualized using ImmunoStar Kit (Wako Pure Chemical).

FACS Analysis.
Staining and flow-cytometric analysis of lung cancer cells were conducted by standard procedures, as described previously (10) , using a FACScan (Becton Dickinson, Mountain View, CA). Briefly, cells (2 x 105) were incubated with specific mAbs and subsequent phycoerythrin-conjugated CD54 (ICAM-1) mAbs at saturating concentrations in FACS medium consisting of HBSS (Nissui, Tokyo), 0.5% HSA, and 0.2% NaN3 (Sigma Aldrich) for 30 min at 4°C. After three washes in FACS medium, cells were analyzed with FACScan. Amplification of the mAb binding was provided by a three-decade logarithmic amplifier. Quantification of the cell surface antigens on one cell was performed using beads, QIFKIT (Dako Japan, Kyoto, Japan) as reported previously (18) . Briefly, five populations of calibration beads, bearing different but well-defined numbers of mAb molecules, were analyzed by FACScan. Mean fluorescence intensity (MFI) of each population of beads were used for construction of the calibration curve. The cell specimen was analyzed by FACScan, and antigen density was calculated by interpolation on calibration curve.

ELISA of sICAM-1.
After treatment of A904L cells with anti-ß1 integrin mAb, mAb13, for 30 min, the cells were washed with PBS twice and were further incubated in RPMI 1640 without FCS for 6 or 24 h. The conditioned medium from the culture was collected and the quantity of sICAM-1 in the medium was determined with sICAM-1 ELISA Kit (Endogen, Woburn, MA), according to the protocol provided by the manufacturer. Each sample was assayed in duplicate.

Adhesion Assay.
The adhesion assay was performed as described previously (17) . Briefly, lung cancer cells were applied to 48-well culture plates (Nunc, Roskilde, Denmark) and cultured in DMEM (Life Technologies) with 10% heat-inactivated FCS. T cells were labeled with sodium 51Cr (DuPont NEN, Wilmington, DE). A total of 1 x 106 T cells, stimulated with 1 µM PMA (Sigma Aldrich), were added to culture plates precoated with lung cancer cells with or without relevant adhesion-blocking mAb (10 µg/ml). All of the mAbs were used at a saturating concentration of 10 µg/ml, which was shown in previous studies to produce a maximum inhibition of the relevant adhesive interaction (18) . After a settling phase of 30 min at 4°C, which also allowed mAb binding, the plates were rapidly warmed to 37°C for 30 min, followed by gentle washing twice with RPMI 1640 at room temperature to completely remove nonadherent T cells. The contents of each well containing adherent T cells were lysed with 250 µl of 1% Triton X-100 (Sigma Aldrich), and {gamma}-emissions of well contents were determined. Data were expressed as mean percentage of binding of indicated cells from a representative experiment.

Cytotoxic Assay.
The cytotoxicity of CTLs against tumor cells was determined by a standard 51Cr-release cytotoxicity assay as described previously (28) . Tumor targets were labeled with sodium 51Cr for 1 h at 37°C and washed. Target cells (5 x 103) were incubated with effector T cells (effector:target ratio, 1:1 to 30:1) in 200 µl of culture medium in a 96-well round-bottomed microtiter plate (Nunc) for 4 h at 37°C. The supernatant (100 µl) was collected, and the samples were counted in a {gamma}-counter. The percent-specific lysis was calculated using the formula:


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
High Expression of ß1 Integrin and ICAM-1 on Lung Cancer Cell Clones.
Initially, we assessed the expression of various cell surface functional molecules on 11 lung cancer cell clones using FACScan. Table 1Citation shows the number of six representative molecules including ICAM-1 and ß1 integrin on each type of cell. Among the screened molecules, ß1 integrin was highly expressed on all of the 11 lung cancer cell clones, with the highest expression of ß1 integrin on A110L (adenocarcinoma), followed by B1203L (squamous cell carcinoma) and A904L (large cell carcinoma). The level of ß1 integrin on the cell surface was independent of the differentiation pattern of the cell clones. ICAM-1 was also highly expressed on 6 of 11 lung cancer cell clones, and the number of ICAM-1 on A110L and A904L exceeded 1 x 106 molecules/cell. The expression was also independent of the differentiation pattern of cancer cells. In contrast, the expression of VCAM-1 and LFA-1 was marginal on all of the 11 clones. We, therefore, assumed that ß1 integrin and ICAM-1 might play an important role in lung cancer cells. To investigate the functional significance of these molecules, we mainly used A904L cell clone in the following experiments, because they highly expressed both ß1 integrin and ICAM-1 as evident in the histogram shown in Fig. 1Citation .


View this table:
[in this window]
[in a new window]

  		Table 1 Phenotypic analysis of multiple lung cancer cell lines

 


View larger version (26K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 1. Histogram of various cell surface molecules expressed on A904L cells. A904L cells were stained with CD29 mAb (MAR4), VCAM-1 mAb (2G7), CD11a mAb (TS1/22), ICAM-1 mAb (HA58), CD44 mAb (NIH 44–1), and Fas mAb (UB2) and flow cytometric analyses were performed using FACScan. Shadows, profiles of the murine IgG used as a negative control. The ordinate, the number of cells stained with the indicated mAb in each logarithmic scale of fluorescence amplifier. Representative data of five similar experiments are shown.

 
Stimulation of ß1 Integrin Down-Regulates ICAM-1 Expression on Lung Cancer Cells.
To characterize the function of ß1 integrin on lung cancer cells, we assessed the effects of stimulation of ß1 integrin on the expression of various cell surface molecules using a specific mAb. Among the screened molecules, flow cytometric analysis showed that the expression of ICAM-1 was markedly reduced by 24-h stimulation of ß1 integrin with mAb13, a specific mAb, on lung cancer cell clone A904L. Stimulation of A904L cells with other anti-ß1 integrin mAbs, including TS2/16, Lia1/2, and MAR4, also down-regulated ICAM-1 expression on the cells (data not shown). In contrast, the expression of VCAM-1, Fas, and MHC class I antigen did not change by stimulation of ß1 integrin with such mAbs (Fig. 2A(a)Citation . However, stimulation of ß1 integrin on the cells failed to change the expression of ICAM-1 on some of the tested cells including B1203L cells, which suggests that some of the lung cancer cells lacked the regulatory mechanism of ICAM-1 expression by ß1 integrin-mediated signaling (Fig. 2A(b)Citation . Also, stimulation of irrelevant mAbs, including CD44 mAb, VCAM-1 mAb, and anti-MHC class I antigen mAb, failed to reduce ICAM-1 expression on A904L cells, even in a higher concentration, 100 µg/ml (Fig. 2BCitation and data not shown, in part). Time course experiments showed that ICAM-1 expression on A110L cells and A904L cells reached minimum levels within 24 h of ß1 integrin stimulation (Fig. 2C)Citation . These results indicate that stimulation of ß1 integrin markedly reduces the expression of ICAM-1 on A904L lung cancer cells, although such expression does not occur without stimulation.

Engagement of ß1 Integrin by Ligand Matrix Glycoproteins Reduces Expression of ICAM-1 on Lung Cancer Cells.
Fibronectin and type I collagen are major ligands for cell surface ß1 integrin (1, 2, 3) . We next assessed the biological activities of fibronectin and type I collagen on the expression of ICAM-1 on A904L cells. Expression of ICAM-1 was markedly reduced by incubation of these cells on fibronectin- or collagen-coated plastic plates for 24 h. In contrast, no change was noted when these cells were incubated in HSA-coated plates (Fig. 3)Citation . These data suggest that fibronectin and type I collagen are possible ligands involved in ß1 integrin-induced reduction of ICAM-1 expression on the surface of A904L lung cancer cells.



View larger version (33K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 3. Reduction of ICAM-1 by adhesion of ß1 integrins to ligand matrix proteins on A904L cells. A904L cells were incubated on plastic plates which were precoated with or without HSA, fibronectin, or type I collagen (10 µg/ml) for 24 h and the expression of ICAM-1 was analyzed by FACScan. Each bar, the number of molecules expressed per cell, calculated using standard QIFKIT beads. Representative data of five similar experiments are shown.

 
ß1 Stimulation Induces Production of sICAM-1 from A904L Cells.
sICAM-1 is known to be released from the cell surface through proteolytic cleavage (36) . Elevated serum levels of sICAM-1 have also been reported in patients with malignant disorders, including malignant melanoma (37) . When A904L cells were stimulated with anti-ß1 mAb mAb13 for 24 h, the levels of sICAM-1 in the conditioned medium of cells was markedly increased, compared with spontaneous secretion of sICAM-1 (Fig. 4A)Citation . In contrast, stimulation of VCAM-1, CD44, or MHC class I antigens by specific mAbs failed to induce the sICAM-1 in the conditioned medium on A904L cells (Fig. 4B)Citation . These results suggest that engagement of ß1 stimulation induces the production of sICAM-1 from A904L cells, which leads to the reduced expression of ICAM-1 on the cell surface.



View larger version (12K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 4. Engagement of ß1 integrins on the production of sICAM-1 from A904L cells. A, sICAM-1 secretion from A904L cells stimulated with ({blacksquare}) or without ({square}) anti-ß1 integrin mAb mAb13 for the indicated duration. B, sICAM-1 secretion from A904L cells with or without the indicated mAbs. sICAM-1 in the conditioned medium was measured by specific ELISA. Representative data of five similar experiments are shown.

 
Involvement of Tyrosine Kinases in ß1 Integrin-induced Down-Regulation of ICAM-1 Expression on A904L Cells.
Signals from integrin receptors are mediated by tyrosine phoshorylation in various cells (38, 39, 40) . To determine the involvement of tyrosine kinases in ß1 integrin-induced reduction of ICAM-1 expression on A904L cells, we pretreated the cells with or without different concentrations of various inhibitors of intracytoplasmic signaling, and ICAM-1 expression was determined by FACScan (Fig. 5)Citation . Pretreatment of cells with tyrosine kinase inhibitors, herbimycin A or genistein, completely inhibited ß1-induced down-regulation of ICAM-1 expression on A904L cells. In contrast, H7 or staurosporine, C-kinase inhibitors, and wortmannin, a PI-3 kinase inhibitor, did not influence ß1 integrin-induced reduction of ICAM-1 expression. These results suggest that ß1 integrin-induced reduction of ICAM-1 expression on A904L cells is mainly mediated through tyrosine kinases.



View larger version (13K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 5. Effects of various signaling inhibitors on ß1 integrin-induced down-regulation of ICAM-1 expression on A904L cells. A904L cells were pretreated with or without indicated concentration of various inhibitors of intracytoplasmic signaling, and ICAM-1 expression was determined by FACScan. Data are expressed as mean percentage of blocking of ICAM-1 expression on A904L cells. Representative data of five similar experiments are shown.

 
Phosphorylation of FAK by ß1 Integrin-mediated Signaling on A904L Cells.
FAK is an important mediator of the integrin signaling induced by adhesion to extracellular matrix proteins (34 , 41) . The expression levels of FAK in A904L cells, A110L cells, and B1203L cells were assessed by Western blotting using anti-FAK Abs (Fig. 6A)Citation . A high level of FAK expression was spontaneously observed in A904L cells and A110L cells, whereas only marginal expression of FAK was seen in B1203L cells. The results suggest that ß1 integrin-mediated signaling could depend on the presence of FAK in the cells. We next examined the effects of ß1 integrin-mediated signaling on the phosphorylation of FAK using anti-phosphorylated tyrosine (FAK, pY397)-specific Ab. Stimulation of A904L cells with anti-ß1 integrin mAb mAb13 markedly induced phosphorylation of FAK in the cells. However, engagement of ß1 integrin with the mAb did not induce tyrosine phosphorylation of FAK in the cells expressing VSV-FAT and VSV-FRNK, a dominant negative truncation of FAK, but ß1 integrin-stimulation induced tyrosine phosphorylation of FAK in the cells expressing VSV-FAK, a wild-type FAK (Fig. 6B)Citation .



View larger version (12K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 6. Expression and tyrosine phosphorylation of FAK in lung cancer cell clones. A, the expression of FAK in A904L cells, A110L cells, and B1203L cells. The expression levels of endogenous FAK in the indicated cells were assessed by Western blotting using anti-FAK Ab. B, the effects of ß1 integrin-mediated signaling on the phosphorylation of FAK in A904L cells expressing with or without FAK-mutants. The tyrosine phos phor y lation of FAK was detected using anti-phosphorylated tyrosine (FAK, pY397)-specific Ab in A904L cells or A904L cells expressing VSV-FAT and VSV-FRNK, a dominant negative truncation of FAK, or VSV-FAK, a wild-type FAK, in the presence or absence of stimulation with anti-ß1 integrin mAb mAb13.

 
Involvement of FAK in ß1 Integrin-induced Reduction of ICAM-1 Expression on A904L Cells.
As described, ICAM-1 expression was markedly decreased by the stimulation of ß1 integrins using specific mAb on A904L cells and also on A904L cells expressing VSV-FAK, a wild-type FAK. However, such a reduction of ICAM-1 expression by the stimulation with anti-ß1 integrin-specific mAb was not observed on the cells expressing VSV-FAT and VSV-FRNK, a dominant negative truncation of FAK (Fig. 7)Citation . These results indicate that phosphorylation of tyrosine residues of FAK is involved in signaling through ß1 integrin-stimulation, which leads to the down-regulation of ICAM-1 expression on lung cancer cells.



View larger version (24K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 7. ß1 integrin-induced down-regulation of ICAM-expression is mediated by FAK on A904L cells. ß1 integrin-stimulated A904L cells, transfected with or without control vector or expression vectors encoding FAK, FAT, or FRNK were analyzed for expression of ICAM-1 using flow cytometry. Data represents the mean number of molecules expressed per cell, calculated using standard QIFKIT beads, in three replicate wells. Representative data of five similar experiments are shown.

 
ß1 Integrin Stimulation Reduces LFA-1/ICAM-1-mediated Adhesion of A904L Cells to T Cells through Tyrosine Kinases.
We next investigated whether ß1 integrin-induced down-regulation of ICAM-1 expression on A904L cells influenced their adhesion to T cells. A904L cells spontaneously adhered to PMA-stimulated T cells and such adhesion was significantly inhibited by anti-LFA-1 mAb, but not by anti-CD44 mAb, indicating that T cell adhesion to A904L cells was mediated by LFA-1/ICAM-1 adhesive pathway (Fig. 8A)Citation . ß1 integrin-induced reduction of adhesion of A904L cells to T cells was completely abrogated by pretreatment of cells with tyrosine kinase inhibitors, herbimycin A or genistein, and such inhibition was concentration-dependent, whereas C-kinase inhibitor, H7, failed to influence cell adhesion (Fig. 8B)Citation . These data suggest that ß1 integrin-induced reduction of adhesion of A904L cells to T cells through LFA-1/ICAM-1 pathway is mediated by tyrosine kinases. Although the reduction of ICAM-1-mediated adhesion of A904L cells, induced by ß1-stimulation, did not change in A904L cells that expressed a wild-type FAK, it was completely inhibited on A904L cells expressing FAT or FRNK, namely dominant negative truncation of FAK (Fig. 8C)Citation . These results indicate that FAK is involved in tyrosine kinase-mediated signaling through ß1 integrin stimulation, which leads to the down-regulation of ICAM-1 expression and subsequent ICAM-1-dependent adhesion of lung cancer cells.



View larger version (27K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 8. ß1 integrin-induced inhibition of LFA-1/ICAM-1-mediated adhesion of A904L cells to T cells through tyrosine kinases. A, adhesion assay of ß1 integrin-stimulated A904L cells to PMA-activated T cells. A904L cells were incubated with or without ß1 mAb mAb13 for 24 h, and 51Cr-labeled T cells were added to the culture in the presence or absence of the indicated blocking mAbs (10 µg/ml), anti-LFA-1 mAb TS1/22 or control CD44 mAb NIH44–1, at 37°C for 30 min. After washing out nonadherent T cells, {gamma}-emissions of adherent cells were counted. B, tyrosine kinase inhibitors result in recovery of ICAM-1-mediated adhesion of A904L cells reduced by ß1 integrin. A904L cells were pretreated with or without indicated concentrations of inhibitors of intracytoplasmic signaling. Adhesion of cells to PMA-activated T cells was assessed as described in "Materials and Methods." C, expression of dominant negative mutation of FAK on A904L cells with restored ICAM-1-mediated adhesion originally reduced by ß1 integrin. A904L cells, expressing FAK, FAT, or FRNK, were incubated with ß1 mAb mAb13 for 24 h, and adhesion of the cells to PMA-activated T cells was assessed as described in "Materials and Methods." Data are expressed as the mean percentage of T cell-adhesion to A904L cells in three replicate wells from one representative experiment among five.

 
Stimulation of ß1 Integrin Influences Killing of Lung Cancer Cells by Autologous CTLs.
Finally, we assessed the response of ß1 integrin-stimulated lung cancer cells to CTL killing. Autologous CTLs, A904L CTLs and B1203L CTLs, derived from patients with A904L cells and B1203L cells, respectively, were examined for autologous cytotoxicity. Cytotoxic activities of A904L CTLs and B1203L CTLs against the respective cancer cells decreased after the addition of anti-LFA-1 mAb and anti-MHC class I mAb, but not anti-MHC class II mAb (Fig. 9)Citation , which indicated that CTL killing activity depends on LFA-1/ICAM-1-mediated adhesion of lung cancer cells to CTLs. When A904L cells were stimulated with ß1 mAb, autologous cytotoxicity by CTLs was reduced, whereas ß1 integrin-stimulation of B1203L cells neither changed ICAM-1 expression on the cells (Fig. 2A)Citation nor their killing by autologous CTLs (Fig. 9)Citation . These results indicated that activation of ß1 integrin on A904L lung cancer cells by cell binding to extracellular matrix leads to escape from autologous CTL killing through ICAM-1 down-regulation and LFA-1/ICAM-1-mediated adhesion of the cells.



View larger version (12K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 9. ß1 integrin stimulation prevents killing of lung cancer cells by autologous CTLs. Autologous CTLs (A904L CTLs and B1203L CTLs) were induced in patients with A904L cells (A) and B1203L cells (B), respectively. A904L cells or B1203L cells (target) were incubated with ({blacksquare}) or without ({square}) ß1 mAb mAb13 for 24 h. After labeling cells with 51Cr, CTLs (effector) derived from patients were added to the culture in the presence or absence of the indicated blocking mAbs (10 µg/ml), anti-LFA-1 mAb TS1/22, or MHC class I mAb W6/32, or MHC class II mAb L227 at 37°C for 4 h. Autologous CTL cytotoxic activity against tumor target cells was assessed by 51Cr-release from target cells stimulated by effector CTL at an effector:target ratio of 30:1. Data are expressed as the mean percentage of 51Cr-release from targets in one representative experiment among five.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Identification of the molecular basis of cellular adhesion and its importance in cell-cell and cell-matrix interactions has progressed during the last decade. The expression and function of adhesion molecules are tightly regulated by several cellular stimuli. Cytokines are potent inducers of adhesion molecule expression. Among these, IL-1ß and tumor necrosis factor {alpha} contribute to the up-regulation of immunoglobulin-superfamily adhesion molecules, such as ICAM-1 and VCAM-1 (23 , 24) . However, based on the results of the present study, we propose a new concept, that stimulation of adhesion molecule ß1 integrin per se plays a pivotal role in the regulation of ICAM-1 expression and ICAM-1-mediated adhesion and/or killing of lung cancer cells. This conclusion is based on the following novel findings: (a) engagement of ß1 integrins on lung cancer cells by a specific mAb as well as ligand matrices such as fibronectin and collagen markedly reduced ICAM-1 expression on the surface, and up-regulated sICAM-1 in the conditioned medium; (b) down-regulation of ICAM-1 induced by ß1 integrin-stimulation was mediated by the signaling pathway of tyrosine kinase, especially involved by FAK; (c) engagement of ß1 integrin also reduced ICAM-1-dependent adhesion of lung cancer cells to T cells, which was completely abrogated by inhibitors of tyrosine kinases and transfection of dominant negative forms of FAK; and (d) stimulation of ß1 integrin prevented lung cancer cells from being killed by autologous CTLs. Therefore, we further propose that ß1 integrin-dependent adhesion to matrix proteins in lung cancer cells can transduce signaling to reduce ICAM-1-expression, ICAM-1-mediated adhesion to CTLs, and the killing of tumor cells by CTLs through tyrosine kinase involved by FAK.

ß1 integrins attach the cell to extracellular matrices or to other cells, and after its ligation, the integrins are found in focal adhesion plaques in which various cytoskeletal proteins accumulate. Engagement of ß1 integrins leads to initiation of intracellular signal transduction, designated "outside to in signaling" through accumulated cytoskeletal signaling kinases, which results in activation, differentiation, development, and mobility of various type of cells including tumor cells (11 , 12) . Several studies have established that among various cytoskeletal proteins, FAK, a cytoplasmic protein tyrosine kinase that localizes focal adhesions, is an important mediator of integrin-mediated signaling and that FAK levels are increased in proliferating cells or advanced tumor cells. Localization of FAK at focal adhesions and its enhanced tyrosine phosphorylation in response to integrin-ligation play a pivotal role in the integrin-mediated signaling cascade in tumor cells (38, 39, 40) . The present study demonstrated that engagement of ß1 integrin markedly down-regulated ICAM-1 expression and subsequent ICAM-1/LFA-1-mediated adhesion of lung cancer cells to T cells. However, ß1 integrin-induced reduction of ICAM-1 expression and ICAM-1-dependent adhesion of the cancer cells were completely inhibited when the cells were pretreated with tyrosine kinase inhibitors, herbimycin A or genistein, but not with C-kinase inhibitors, H7 and staurosporine, nor a PI-3 kinase inhibitor, wortmannin. Furthermore, it is noteworthy that ß1 integrin-induced down-regulation of ICAM-1 expression and ICAM-1-dependent adhesion of lung cancer cells expressing FRNK or FAT, dominant negative truncations of FAK, were completely inhibited, whereas the reduction of ICAM-1 expression and ICAM-1-dependent adhesion did not change in cells expressing a wild-type FAK. These findings suggest that ß1 integrin induced down-regulation of ICAM-1 expression and subsequent ICAM-1-dependent adhesion of lung cancer cells to T cells, which was brought about by ß1 integrin-mediated "outside to in" signaling of tyrosine kinases, specifically involve FAK.

ICAM-1 belongs to the immunoglobulin superfamily, and its expression is highly inducible on various cells during inflammation and in response to proinflammatory cytokines through inside to out signaling (23 , 24 , 42, 43, 44) . To our knowledge, our study is the first to demonstrate that expression of ICAM-1 on lung cancer cells is markedly down-regulated by signaling induced by the ligation of ß1 integrin. The role of ICAM-1 in immune vigilance against tumor cells is still to be defined. Several studies have suggested that the induction of ICAM-1 molecules on tumor cells results in enhanced adhesion of these cells to CTLs or natural killer cells through a LFA-1/ICAM-1 pathway and their subsequent killing (45, 46, 47) . In fact, overexpression of ICAM-1 is associated with a favorable prognosis in tumors including non-small cell lung carcinoma (48) . However, although various tumor cells are known to express ICAM-1 in vitro, many tumor cells escape from CTL-induced killing in vivo. In the present study, reduced expression of ICAM-1 and ICAM-1-mediated adhesion of the lung cancer cells to T cells induced by engagement of ß1 integrin resulted in blocking of killing of cancer cells by autologous CTLs. In tumor tissues, cancer cells, including lung cancer cells, are surrounded by extracellular matrix proteins such as fibronectin and collagen, mainly through their receptors, ß1 integrins on the cells. Hence, engagement of ß1 integrins by matrix proteins always occurs in cancer cells and continuous stimulation of these cells via ß1 integrin should reduce ICAM-1-expression, ICAM-1-mediated adhesion to CTL and ICAM-1-mediated killing by CTLs, which leads to escape from CTL-mediated killing of lung cancer cells in vivo. Although the promise of cancer treatment through manipulation of tumor immunology has yet to be fulfilled, it is noteworthy that several likely mechanisms through which tumors escape immune surveillance have been unraveled at a molecular level only in recent years.

On the basis of the findings of the present study, we propose that stimulation of adhesion molecules regulates expression of other adhesion molecules on the same cell, e.g., stimulation of ß1 integrin by cross-linking or ligation with matrix proteins reduces ICAM-1 expression. We also propose that engagement of ß1 integrin on lung cancer cells by matrix proteins plays a role in the reduction of ICAM-1-expression, ICAM-1-mediated adhesion to CTLs and killing by CTLs, resulting in the prevention of cancer cells being killed by CTLs. Our findings warrant additional studies on the molecular mechanisms involved in the regulation of ß1 integrin-mediated signaling. Such studies would enhance our understanding of the complex processes involved in tumor progression and metastasis and allow the design of new therapeutic approaches to control such processes.


   ACKNOWLEDGMENTS
 
We thank Ms. T. Adachi for excellent technical assistance. We also thank Drs. K. M. Yamada, W. Newman, and S. Shaw for providing mAbs and reagents, and Dr. F. G. Issa (Word-Medex, Sydney, Australia) for the careful reading and editing of the manuscript.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan. Back

2 To whom requests for reprints should be addressed, at The First Department of Internal Medicine, University of Occupational and Environmental Health, Japan, School of Medicine, 1-1 Iseigaoka, Yahatanishi-ku, Kitakyushu 807-8555, Japan. Phone: 81-93-603-1611, extension 2426; Fax: 81-93-691-9334; E-mail: tanaka{at}med.uoeh-u.ac.jp Back

3 The abbreviations used are: FAK, focal adhesion kinase; FAT, focal adhesion targeting domain; FRNK, FAK-related nonkinase; ICAM-1, intercellular adhesion molecule-1; LFA-1, leukocyte function-associated antigen-1; Ab, antibody; mAb, monoclonal Ab; PI, phosphatidylinositol; RLNL, regional lymph node lymphocyte; FACS, fluorescence-activated cell sorting; IL, interleukin; HSA, human serum albumin; sICAM-1, soluble ICAM-1; PMA, phorbol 12-myristate 13-acetate. Back

Received 1/26/00. Accepted 12/27/00.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Nicolson G. L. Tumor cell instability, diversification, and progression to the metastatic phenotype: from oncogene to oncofetal expression. Cancer Res., 47: 1473-1487, 1987.[Abstract/Free Full Text]
  2. Hart I. R. "Seed and Soil" revisited: mechanisms of site-specific metastasis. Cancer Metastasis Rev., 1: 5-16, 1982.[Medline]
  3. Liotta L. A., Rao C. N., Barsky S. H. Tumor invasion and the extracellular matrix. Lab. Investig., 49: 636-649, 1983.[Medline]
  4. Humphries M. J., Olden K., Yamada K. M. A synthetic peptide fibronectin inhibits experimental metastasis of murine melanoma cells. Science (Washington DC), 233: 467-470, 1986.[Abstract/Free Full Text]
  5. McCarthy J. B., Furcht L. T. Laminin and fibronectin promote the haptotactic migration of B16 mouse melanoma cells in vitro. J. Cell Biol., 98: 1474-1480, 1984.[Abstract/Free Full Text]
  6. Pasqualini R., Bourdoulous S., Koivunen E., Woods V. L., Jr., Ruoslahti E. A polymeric form of fibronectin has antimetastatic effects against multiple tumor types. Nat. Med., 2: 1197-1203, 1996.[Medline]
  7. Sethi T., Rintoul R. C., Moore S. M., MacKinnon A. C., Salter D., Choo C., Chilvers E. R., Dransfield I., Donnelly S. C., Strieter R., Haslett C. Extracellular matrix proteins protect small cell lung cancer cells against apoptosis: a mechanism for small cell lung cancer growth and drug resistance in vivo. Nat. Med., 5: 662-668, 1999.[Medline]
  8. Schwartz R. H. Models of T cell anergy: is there a common molecular mechanism?. J. Exp. Med., 184: 1-8, 1996.[Free Full Text]
  9. Kumar C. C. Signaling by integrin receptors. Oncogene, 17: 1365-1373, 1998.[Medline]
  10. Tanaka Y., Albelda S. M., Horgan K. J., van Seventer G. A., Shimizu Y., Newman W., Hallam J., Newman P. J., Buck C. A., Shaw S. CD31 expressed on distinctive T cell subsets is a preferential amplifier of ß1 integrin-mediated adhesion. J. Exp. Med., 176: 245-253, 1992.[Abstract/Free Full Text]
  11. Ruoslahti E. Integrins. J Clin Investig., 87: 1-5, 1991.
  12. Hynes R. O. Integrins: versatility, modulation, and signaling in cell adhesion. Cell, 69: 11-25, 1992.[Medline]
  13. Plantefaber L. C., Hynes R. O. Changes in integrin receptors on oncogenically transformed cells. Cell, 56: 281-290, 1989.[Medline]
  14. Giancotti F. G., Mainiero F. Integrin-mediated adhesion and signaling in tumorigenesis. Biochim. Biophys. Acta., 1198: 47-64, 1994.[Medline]
  15. Nip J., Rabbani S. A., Shibata H. R., Brodt P. Coordinated expression of the vitronectin receptor and the urokinase-type plasminogen activator receptor in metastatic melanoma cells. J. Clin. Investig., 95: 2096-2103, 1995.
  16. Juliano R. L. The role of ß1 integrins in tumors. Semin. Cancer Biol., 4: 277-283, 1993.[Medline]
  17. Tanaka Y., Kimata K., Wake A., Mine S., Morimoto I., Yamakawa N., Habuchi H., Ashikari S., Yamamoto H., Sakurai K., Yoshida K., Suzuki S., Eto S. Heparan sulfate proteoglycan on leukemic cells is primarily involved in integrin triggering and its mediated adhesion to endothelial cells. J. Exp. Med., 184: 1987-1997, 1996.[Abstract/Free Full Text]
  18. Tanaka Y., Mine S., Hanagiri T., Hiraga T., Morimoto I., Figdor C. G., van Kooyk Y., Nakamura T., Yasumoto K., Eto S. Constitutive up-regulation of integrin-mediated adhesion of tumor-infiltrating lymphocytes to osteoblasts and bone marrow-derived stromal cells. Cancer Res., 58: 4138-4145, 1998.[Abstract/Free Full Text]
  19. Gismondi A., Milella M., Palmieri G., Piccoli M., Frati L., Santoni A. Stimulation of protein tyrosine phosphorylation by interaction of NK cells with fibronectin via {alpha}4ß1 or {alpha}5ß1. J. Immunol., 154: 3128-3137, 1995.[Abstract]
  20. Freedman A. S., Rhynhart K., Nojima Y., Svahn J., Eliseo L., Benjamin C. D., Morimoto C., Vivier E. Stimulation of protein tyrosine phosphorylation in human B cells after ligation of the ß1 integrin VLA-4. J. Immunol., 150: 1645-1652, 1993.[Abstract]
  21. Nojima Y., Rothstein D. M., Sugita K., Schlossman S. F., Morimoto C. Ligation of VLA-4 on T cells stimulates tyrosine phosphorylation of a 105-kD protein. J. Exp. Med., 175: 1045-1053, 1992.[Abstract/Free Full Text]
  22. Burridge K., Fath K., Kelly T., Nuckolls G., Turner C. Focal adhesions: transmembrane junctions between the extracellular matrix and the cytoskeleton. Annu. Rev. Cell. Biol., 4: 487-525, 1988.
  23. Sallusto F., Lanzavecchia A. Efficient presentation of soluble antigen by cultured human dendritic cells is maintained by GM-CSF plus IL-4 and downregulated by TNF-{alpha}. J. Exp. Med., 179: 1109-1118, 1994.[Abstract/Free Full Text]
  24. Shen J., Devery J. M., King N. J. Adherence status regulates the primary cellular activation responses to the Flavivirus West Nile. Immunology, 84: 254-264, 1995.[Medline]
  25. Dustin M. L., Springer T. A. T-cell receptor cross-linking transiently stimulates adhesiveness through LFA-1. Nature (Lond.), 341: 619-624, 1989.[Medline]
  26. Mukai S., Kagamu H., Shu S., Plautz G. E. Critical role of CD11a (LFA-1) in therapeutic efficacy of systemically transferred antitumor effector T cells. Cell. Immunol., 192: 122-132, 1999.[Medline]
  27. Yasumoto K., Takeo S., Yano T., Nakahashi H., Nagashima A., Sugimachi K., Nomoto K. Role of tumor infiltrating lymphocytes in the host defense mechanism against lung cancer. J. Surg. Oncol., 38: 221-226, 1988.[Medline]
  28. Yoshino I., Yano T., Murata M., Ishida T., Sugimachi K., Kimura G., Nomoto K. Tumor-reactive T-cells accumulate in lung cancer tissues but fail to respond due to tumor cell derived factor. Cancer Res., 47: 775-781, 1992.
  29. Yasumoto K., Ohta O., Nomoto K. Cytotoxic activity of carcinoma cells in patients with lung cancer. Gann, 67: 505-511, 1976.[Medline]
  30. Yoshino I., Takenoyama M., Fujie H., Hanagiri T., Yoshimatsu T., Imahayashi S., Eifuku R., Ogami A., Yano K., Osaki T., Nakanishi R., Ichiyoshi Y., Nomoto K., Yasumoto K. The induction of cytotoxic T lymphocytes against HLA-A locus-matched lung adenocarcinoma in patients with non-small cell lung cancer. Jpn. J. Cancer Res., 88: 743-749, 1997.[Medline]
  31. Takenoyama M., Yoshino I., Fujie H., Hanagiri T., Yoshimatsu T., Imahayashi S., Eifuku R., Nomoto K., Yasumoto K. Autologous tumor-specific cytotoxic T lymphocytes in a patient with lung adenocarcinoma: implications of shared antigens expressed in HLA-A24 lung cancer cells. Jpn. J. Cancer Res., 89: 60-66, 1998.[Medline]
  32. Fujita J., Saijo N., Sasaki Y., Futami H., Ishihara J., Takahashi H., Hoshi A., Hamburger A. W. Detection of cytotoxicity of freshly obtained lymphocytes and of lymphocytes activated with recombinant interleukin II (rIL-2) against lung cancer cell lines by human tumor clonogenic assay (HTCA). Eur. J. Cancer Clin. Oncol., 4: 445-450, 1986.
  33. Schoof D. D., Selleck C. M., Massaro A. F., Jung S. E., Eberlein T. J. Activation of human tumor-infiltrating lymphocytes by monoclonal antibodies directed to the CD3 complex. Cancer Res., 50: 1138-1143, 1990.[Abstract/Free Full Text]
  34. Tamura M., Gu J., Matsumoto K., Aota S., Parsons R., Yamada K. M. Inhibition of cell migration, spreading, and focal adhesions by tumor suppressor PTEN. Science (Washington DC), 280: 1614-1617, 1998.[Abstract/Free Full Text]
  35. Gu J., Tamura M., Pankow R., Danen E. H., Takino T., Matsumoto K., Yamada K. M. Shc and, FAK differentially regulate cell motility and directionality modulated by PTEN. J. Cell Biol., 146: 389-403, 1999.[Abstract/Free Full Text]
  36. Champagne B., Tremblay P., Cantin A., St. Pierre Y. Proteolytic cleavage of ICAM-1 by human neutrophil elastase. J. Immunol., 161: 6398-6405, 1998.[Abstract/Free Full Text]
  37. Harning R., Mainolfi E., Bystryn J. C., Henn M., Merluzzi V. J., Rothlein R. Serum levels of circulating intercellular adhesion molecule 1 in human malignant melanoma. Cancer Res., 51: 5003-5005, 1991.[Abstract/Free Full Text]
  38. Clark E. A., Brugge J. S. Integrins and signal transduction pathways: the road taken. Science (Washington DC), 268: 233-239, 1995.[Abstract/Free Full Text]
  39. Parsons J. T. Integrin-mediated signaling: regulation by protein tyrosine kinases and small GTP-binding proteins. Curr. Opin. Cell. Biol., 8: 146-152, 1996.[Medline]
  40. Schwartz M. A., Schaller M. D., Ginsberg M. H. Integrins: emerging paradigms of signal transduction. Annu. Rev. Cell. Dev. Biol., 11: 549-599, 1995.[Medline]
  41. Tamura M., Gu J., Takino T., Yamada K. M. Tumor suppressor PTEN inhibition of cell invasion, migration, and growth: differential involvement of focal adhesion kinase and p130Cas. Cancer Res., 59: 442-449, 1999.[Abstract/Free Full Text]
  42. Simmons D., Makgoba M. W., Seed B. ICAM, an adhesion ligand of LFA-1, is homologous to the neural cell adhesion molecule NCAM. Nature (Lond.), 331: 624-627, 1988.[Medline]
  43. Wawryk S. O., Novotny J. R., Wicks I. P., Wilkinson D., Maher D., Salvaris E., Welch K., Fecondo J., Boyd A. W. The role of the LFA-1/ICAM-1 interaction in human leukocyte homing and adhesion. Immunol. Rev., 108: 135-161, 1989.[Medline]
  44. Seth R., Raymond F. D., Makgoba M. W. Circulating ICAM-1 isoforms: diagnostic prospects for inflammatory and immune disorders. Lancet, 338: 83-84, 1991.[Medline]
  45. Ramsey P. S., Dean P. A., Tasimowicz-Alpini A., Donohue J. H., Nelson H. Interferon-gamma-induced intercellular adhesion molecule-1: expression on human tumor cells enhances bifunctional antibody-mediated lysis through a lymphocyte function-associated antigen-1dependent mechanism. Cancer Res., 53: 4652-4657, 1993.[Abstract/Free Full Text]
  46. Azuma A., Yagita H., Matsuda H., Okamura K., Niitani H. Induction of ICAM-1 on SCLC cell lines by {gamma}-IFN enhances spontaneous and bispecific anti-CD3 x antitumor antibody-directed LAK cell cytotoxicity. Cancer Res., 52: 4890-4894, 1992.[Abstract/Free Full Text]
  47. Schardt C., Heymanns J., Schardt C., Rotsch M., Havemann K. Differential expression of the ICAM-1 in lung cancer cell lines of various histological types. Eur. J. Cancer, 29: 2250-2255, 1993.
  48. Passlick B., Izbicki J. R., Simmel S., Kubuschok B., Karg O., Habekost M., Thetter O., Schweiberer L., Pantel K. Expression of major histocompatibility class I and class II antigens and ICAM-1 on operable NSCLC: frequency and prognostic significance. Eur. J. Cancer, 30: 376-381, 1994.



This article has been cited by other articles:


Home page
 Cancer Res.Home page
A. Hamai, F. Meslin, H. Benlalam, A. Jalil, M. Mehrpour, F. Faure, Y. Lecluse, P. Vielh, M.-F. Avril, C. Robert, et al.
ICAM-1 Has a Critical Role in the Regulation of Metastatic Melanoma Tumor Susceptibility to CTL Lysis by Interfering with PI3K/AKT Pathway
Cancer Res., December 1, 2008; 68(23): 9854 - 9864.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
S. Abouzahr-Rifai, M. Hasmim, H. Boukerche, J. Hamelin, B. Janji, A. Jalil, C. Kieda, F. Mami-Chouaib, J. Bertoglio, and S. Chouaib
Resistance of Tumor Cells to Cytolytic T Lymphocytes Involves Rho-GTPases and Focal Adhesion Kinase Activation
J. Biol. Chem., November 14, 2008; 283(46): 31665 - 31672.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
H. L. Goel, L. Moro, M. King, N. Teider, M. Centrella, T. L. McCarthy, M. Holgado-Madruga, A. J. Wong, E. Marra, and L. R. Languino
{beta}1 Integrins Modulate Cell Adhesion by Regulating Insulin-Like Growth Factor-II Levels in the Microenvironment
Cancer Res., January 1, 2006; 66(1): 331 - 342.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
K. Liu, S. A. Caldwell, and S. I. Abrams
Cooperative Disengagement of Fas and Intercellular Adhesion Molecule-1 Function in Neoplastic Cells Confers Enhanced Colonization Efficiency
Cancer Res., February 1, 2005; 65(3): 1045 - 1054.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
S. Kammerer, R. B. Roth, R. Reneland, G. Marnellos, C. R. Hoyal, N. J. Markward, F. Ebner, M. Kiechle, U. Schwarz-Boeger, L. R. Griffiths, et al.
Large-Scale Association Study Identifies ICAM Gene Region as Breast and Prostate Cancer Susceptibility Locus
Cancer Res., December 15, 2004; 64(24): 8906 - 8910.
[Abstract] [Full Text] [PDF]

Home page
 JCOHome page
H. Endoh, S. Tomida, Y. Yatabe, H. Konishi, H. Osada, K. Tajima, H. Kuwano, T. Takahashi, and T. Mitsudomi
Prognostic Model of Pulmonary Adenocarcinoma by Expression Profiling of Eight Genes As Determined by Quantitative Real-Time Reverse Transcriptase Polymerase Chain Reaction
J. Clin. Oncol., March 1, 2004; 22(5): 811 - 819.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Cancer Res.Home page
E. E. Hanekamp, S. C. J. P. Gielen, E. Smid-Koopman, L. C. M. Kuhne, P. E. de Ruiter, S. Chadha-Ajwani, A. O. Brinkmann, J. A. Grootegoed, C. W. Burger, F. J. Huikeshoven, et al.
Consequences of Loss of Progesterone Receptor Expression in Development of Invasive Endometrial Cancer
Clin. Cancer Res., September 15, 2003; 9(11): 4190 - 4199.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
M. Yasuda, M. Takenoyama, Y. Obata, M. Sugaya, T. So, T. Hanagiri, K. Sugio, and K. Yasumoto
Tumor-infiltrating B Lymphocytes as a Potential Source of Identifying Tumor Antigen in Human Lung Cancer
Cancer Res., March 1, 2002; 62(6): 1751 - 1756.
[Abstract] [Full Text] [PDF]

Home page
 Int ImmunolHome page
M. Yasuda, Y. Tanaka, K. Fujii, and K. Yasumoto
CD44 stimulation down-regulates Fas expression and Fas-mediated apoptosis of lung cancer cells
Int. Immunol., October 1, 2001; 13(10): 1309 - 1319.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Yasuda, M.
Right arrow Articles by Yasumoto, K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Yasuda, M.
Right arrow Articles by Yasumoto, K.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online